Phytopathologia Mediterranea (2014) 53, 3, 438−450 DOI: 10.14601/Phytopathol_Mediterr-14027

RESEARCH PAPERS Molecular analysis of the 3’ terminal region of yellow dwarf from onion in southern Italy

1,2 3 3 1 1 Ariana MANGLLI , Heyam S. MOHAMMED , Adil Ali EL HUSSEIN , Giovanni E. AGOSTEO , Giuliana ALBANESE 2 and Laura TOMASSOLI

1 Dipartimento di Agraria, Università degli Studi Mediterranea di Reggio Calabria, Loc. Feo di Vito, 89122, Reggio Calabria, Italy 2 Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Plant Pathology Research Centre, Via C. G. Bertero 22, 00156, Roma, Italy 3 Department of Botany, Faculty of Science, University of Khartoum, PO Box 321 11115, Khartoum, Sudan

Summary. Onion yellow dwarf virus (OYDV) is an economically important pathogen causing severe disease in gar- lic, onion and other crops. Eleven isolates of OYDV, all from onion originating from Calabria, southern Italy, were genetically analyzed. An OYDV onion isolate from Sudan was also included in this study. The 3’ terminal region of about 2.5 kb of the twelve isolates were sequenced and the sequences comprising a part of the nuclear in- clusion a (NIa-Pro), the complete nuclear inclusion b (NIb) and coat protein (CP) genes and the 3’ untranslated re- gion (3’UTR), were compared to each other and to corresponding sequences of other OYDV isolates from different countries and Allium hosts. The within-population nucleotide identity of the Italian OYDV onion isolates was very high (more than 99.3%), whereas nucleotide identity between them and OYDV onion isolates from Germany was 94%, Argentina 92% and Sudan 87%. Recombination analysis among the complete 3’ terminal sequences showed putative recombination breakpoints in the NIb region of the Argentine isolate, with the minor parent related to the Sudanese isolate. Comparison between OYDV isolates from onion and isolates from produced identities of 77-78% for the complete nucleotide region. When the 3’ terminal nucleotide sequence and the complete NIb pro- tein were analyzed, the phylogenetic analysis generated rooted trees with high bootstrap values (100%), showing a genetic grouping into two well separated clades distinctive for onion and garlic isolates of OYDV. Phylogenetic analysis of CP protein and 3’UTR showed lower bootstrap separation values and no distinct sub-grouping of the OYDV isolates from the two major Allium species.

Key words: OYDV, Allium cepa, molecular characterization, NIb and CP proteins, 3’UTR region.

Introduction hab et al., 2009; Fayad-André et al., 2011; Katis et al., 2012; Kumar et al., 2012; Mohammed et al., 2013). The Onion yellow dwarf virus (OYDV), is one of the virus is reported to be transmitted in a non-persis- most important affectingAllium species (Van tent manner by more than 50 aphid species (Drake et Dijk, 1993). The virus has spread worldwide and a al., 1933), including Myzus persicae (Sulzer), which is high incidence has been found in many countries, the most efficient vector followed by Aphis craccivo- including Greece, Argentina, The Czech Republic, ra (Koch), and A. gossypii (Glover) (Abd El-Wahab, Egypt, Brazil, India and Sudan (Dovas et al., 2001; 2009; Kumar et al., 2011). OYDV was first identified Conci et al., 2003; Klukáčková et al., 2004; Abd El Wa- in onion (Allium cepa L.) in Iowa, USA (Melhus et al., 1929) and has a limited host range restricted to the Al- lium genus (family Amaryllidaceae). It mainly infects Corresponding author: L. Tomassoli onion and garlic (Allium sativum L.), but also shal- E-mail: [email protected] lot (A. ascalonicum L.), leek (A. fistolosum) and other

438 ISSN (print): 0031-9465 www.fupress.com/pm ISSN (online): 1593-2095 © Firenze University Press Molecular variability of Onion yellow dwarf virus from onion

Allium species from Asia. No wild species of Allium was first identified only in southern Italy (Dovas and or other weeds have been reported as natural hosts. Vovlas, 2003; Parrella et al., 2005), without providing The main pathways for OYDV survival are therefore published sequences and phylogenetic analysis of volunteer onion plants from previous crops, onion the onion isolates detected. sets or vernalized bulbs for bulb-to-seed crops (Katis In the present study, we identified OYDV in et al., 2012). southern Italy (Calabria) in the onion cultivar ‘Rossa In onion, OYDV causes yellow striping or yellow- di Tropea’ (protected by the IGP European trade- ing, curling, flattening and crinkling of leaves and mark), and the 3’ terminal sequences of eleven OYDV dwarfing of plants. In seed crops, flower stems re- isolates were determined, including part of the NIa- main turgid and round but show yellowing, distor- Pro gene, the complete nuclear inclusion b (NIb) and tion and curling. In addition, they are shorter than coat protein (CP) genes and 3’ untranslated region normal, and produce smaller flowers with reduced (3’UTR). Nucleotide and amino acid sequences were numbers of seeds that are often of poor quality (Ku- examined and compared with those of three OYDV- mar et al., 2012). OYDV infection causes seed yield O isolates, one from Sudan (included in this study) loss up to 50%, reductions in bulb weight and size up and two from Celli et al. (2013). The sequences were to 40%, and impairs seed storage (Conci et al., 2003; also compared with other OYDV-G isolates reported Elnagar et al., 2011; Kumar et al., 2012). in the GenBank database, to determine the similari- Numerous surveys and epidemiological studies ties and differences between the two viral subgroups regarding the garlic strain of OYDV, named OYDV-G of different host origin. Recombination analysis was (Van Dijk, 1993; Katis et al., 2012) have been carried accomplished among the complete 3’ terminal se- out in many countries where the virus causes severe quences of OYDV-O isolates. damage to garlic and other minor Allium spp. (shal- lot, leek and scallion), frequently found in mixed in- fections with other viral species of the genera Poty- Materials and methods virus, Carlavirus and Allexivirus (Fajardo et al., 2001; Virus isolates Fayad-André et al., 2011; Bagi et al., 2012; Katis et al., 2012; Mohammed et al., 2013). Studies on OYDV in- This study focused on the onion cultivar ‘Rossa fecting onion crops, hereafter designated as OYDV- di Tropea’ (Red of Tropea), grown in the Tropea area O, have also been reported, concerning occurrence, of Calabria (southern Italy), collecting two different distribution and transmission (Hoa et al., 2003; Ku- biotypes of the cultivar characterized by round (in- mar et al., 2011; 2012; Elnagar et al., 2011; Katis et al. dicated as ‘T’) or long (indicated as ‘L’) red bulbs. 2012; Velasquez-Valle et al., 2012; Sevik and Akcura, In the same location, different fields regarding both 2013). bulb (first year: seed-to-bulb) and seed (second OYDV is a 10,538 nucleotide long, single-strand- year: bulb-to-seed) productions were surveyed for ed positive sense RNA virus (GenBank database, OYDV infections during 2012-2013. Samples were Reference accession No: NC005029) of the genus collected from plants showing severe symptoms of (family Potyviridae). The genome encodes yellow striping and crinkling of leaves and distor- a single large polyprotein yielding up to ten mature tion and dwarfing of flower stems (Figure 1). OYDV proteins between the terminal untranslated regions infection was confirmed by DAS-ELISA tests (Loewe (Gibbs and Oshima, 2010), and an overlapping open Biochemica GmbH), and 11 isolates were selected reading frame (ORF), termed pipo, encoding for an for molecular characterization. An OYDV-O isolate eleventh protein (Chung et al., 2008). Information re- (designated O.70) from Sudan, previously identified garding the variability of populations of OYDV has by H.S. Mohammed at Plant Pathology Research been obtained through molecular techniques, and Centre, Rome (Italy), was also included in this study. genetic studies have provided data mainly on garlic isolates, whereas less information is available on on- Viral RNA and RT-PCR assays ion isolates (Arya et al., 2006; Celli et al., 2013). In Italy, the presence of OYDV has long been as- Total RNA was extracted from fresh host leaf tis- sumed, based on symptomathology recorded in on- sue using Real Total RNA from Tissue and Cell kit ion (Marani and Bertaccini, 1983). However, OYDV (Durviz) according to the manufacturer’s instruc-

Vol. 53, No. 3, December, 2014 439 A. Manglli et al.

Figure 1. Flower stems from an onion seed crop showing distortion and curling caused by OYDV.

tions. Primer sets (Table 1) were designed by mul- min, 57°C for 1 min, 72°C for 1 min, and a final ex- tiple nucleotide sequence alignments of OYDV iso- tension at 72°C for 10 min. A genus-specific primer lates from Allium spp. available in GenBank, choos- pair (CPUP/P9502) amplifying the 3’ CP- 3’UTR re- ing highly conserved nucleotide regions within the gion of was also used to complete the species. The annealing positions of the primers were 3’ terminal region of the isolates (Van der Vlugt et arranged in order to obtain contiguous fragments al., 1999) under the following conditions: RT at 46°C with the ends overlapping between each one. For for 60 min, initial denaturation at 94°C for 5 min fol- each primer set, single-step RT-PCR amplification lowed by 35 cycles of 94°C for 2 min, 52°C for 2 min, was performed in a total volume of 25 μL, includ- 72°C for 2 min, and final extension at 72°C for 10 min. ing 2 μL of Total RNA, GoTaq buffer 1× (Promega), 2.5 mM each dNTPS, 4 μM of sense and antisense Cloning and sequencing of PCR products primers, 1.2 U of AMV-RT (Promega), 20 U of RNase Out (Life Technologies) 0.75 U GoTaq Polymerase The PCR fragment of one Italian and the Suda- (Promega). The RT-PCR consisted of a reverse tran- nese OYDV-O isolates were purified using Amicon scription at 46°C for 30 min, initial denaturation at Microcon-PCR Centrifugal Filter Devices (Millipore 95°C for 5 min followed by 35 cycles of 94°C for 1 Corporation), ligated into pGEM-T Easy Vector (Pro-

440 Phytopathologia Mediterranea Molecular variability of Onion yellow dwarf virus from onion

Table 1. Newly designed primers used for NIb and CP gene amplification and sequencing of OYDV isolates.

Nucleotide Amplicon Primer Sequencea (5’ to 3’) Gene positionb size (bp)

OYDV-NIb F1 AACATTGCATGGGGWTCWCT NIa 7825-7844 706 OYDV-NIb R1 CCTTMCCACCAAGYAACG NIb 8530-8513 OYDV-NIB F2 GGCATCTGGAACGGATCTT NIb 8419-8437 754 OYDV-NIb R2 CTATACGTTCCATGTCCAATTTT NIb 9172-9150 OYDV-NIb/CP F1 CATCCAGATCACGAGGGAAT NIb 9001-9020 984 OYDV-NIb/CP R1 TGTGGCATTTCGGTATTCAA CP 9984-9965 OYDV-CP F2 YGTYGAYRCTGGMACHACYG CP 9558-9577 615 OYDV-CP R2 RTTACCATCMARGCCAAACA CP 10172-10154

a IUPAC Code for the mixed bases. b The position of the primers are indicated according to the OYDV genome sequence (JX433020).

mega) and transformed into Escherichia coli strain in nodes was based on 1000 bootstrap replicates. JM109 High Efficiency Competent cells (Promega) Shallot yellow stripe virus (SYSV) was chosen as the according to the manufacturer’s instructions. Plas- out-group (accession No. NC007433) as the closest mid DNA containing an insert of the expected size species among the members of the genus Potyvirus was extracted from three clones for each viral isolate (Gibbs and Ohshima, 2010). Occurrence of recombi- using Quantum Prep Plasmid Miniprep Kit (Bio-Rad nation events amongst OYDV isolates was examined Laboratories). Inserts were sequenced in both direc- using the Recombinant Detection Program (RDP4). tions using T7 and SP6 universal primers (Bio-Fab Research). The amplified products of the other Italian OYDV-O were directly sequenced in both directions. Results The consensus sequence for both cloned and non- Analysis of the complete 3’ terminal region cloned products assembly of the 3’ terminal region of OYDV was compiled using ClustalW implemented The 3’ terminal nucleotide region of all OYDV- in MEGA 5 (Tamura et al., 2011). O isolates was successfully amplified in one-step RT-PCR using the specific primer pairs (Table 1) de- signed from the 3’ terminus of the NIa gene to the 3’ Sequence comparison, phylogenetic and terminus of CP gene and the CPUP/P9502 primer set recombination analysis for amplification of 3’UTR codon. The consensus se- Amino acid (aa) sequences were predicted us- quences of the insert ligated into pGEM-T Easy Vec- ing Translate Expasy (http://www.expasy.org), and tor from the three clones considered for the Italian pairwise and multiple alignments of all nt and aa se- 152T and the Sudanese O.70 isolates, were identical. quence data sets, according to each genomic coding The 3’ terminal nucleotide sequences of all isolates and uncoding region and the complete 2637 nt se- were deposited in the GenBank database (Table 2). quences, were performed by both EMBOSS-Needle The analysis of the obtained sequences revealed a (http://www.ebi.ac.uk) and ClustalW2 (MEGA 5). single ORF of 2427 nt encoding a polyprotein which Phylogenetic and evolutionary relationships among included the C-terminal of the NIa-Pro protein and OYDV isolates were examined by generating phylo- the complete NIb and CP proteins. The ORF was fol- genetic rooted trees of both nt and aa sequences using lowed by an UTR of 210 nucleotides excluding the the neighbour–joining (NJ) method and the Kimura poly (A) tail. two-parameter model implemented in MEGA 5 (Ta- The pairwise sequence alignment of the Italian mura et al., 2011). Evaluation of statistical confidence OYDV-O isolates revealed nucleotide identity of up

Vol. 53, No. 3, December, 2014 441 A. Manglli et al. to 99.9% with each other, and up to 94.2% with the the Sudanese isolate (KF623541). The nucleotide isolate from Germany (JX433020), 91.9% with the identity ranged from 76.7 to 78.3% when all OYDV- isolate from Argentina (JX433019) and 87.1% with O isolates were compared with the corresponding sequence of OYDV-G isolates retrieved from the GenBank (Table 3). The partial NIa Pro-CP region of all the onion iso- Table 2. Identification code and GenBank accession num- bers of the OYDV-O isolates from the Tropea area in lates produced a predicted polyprotein of 809 amino Calabria (southern Italy) and the OYDV-O isolate from acids with an identity of more than 99.1% amongst Sudan. Italian isolates, and average identity values between the Italian OYDV-O population and the Argentina Isolatea Accession No. isolate of 97.5%, the German isolate of 97.2% and the Sudanese isolate of 94.5%. The pairwise alignments 152T KF623530 of aa sequences of the Italian onion isolates revealed 154T KF623531 an average identity of 86.2% with the corresponding sequences of garlic isolates. 155T KF623532 The Sudanese isolate showed an amino acid iden- 9L.Se KF623533 tity of 96.3% with the corresponding NIa Pro-CP of 11L.Se KF623534 the Argentine onion isolate, 93.6% the German onion isolate and from 85.3% to 86.4%, with all OYDV gar- 5L KF623535 lic isolates retrieved from GenBank. 8T KF623536 The polyprotein of Italian isolates, Sudanese iso- 6L.Se KF623537 late and of two already published OYDV-O isolates (Celli et al., 2013) showed conserved cleavage sites 13L.Se KF623538 (Adams et al., 2005a) at the NIa-Pro/NIb (SFQ/SSE) 2T.Se KF623539 and the NIb/CP (RYQ/AGP). When OYDV-O were 27T.Se KF623540 compared with OYDV-G isolates, the NIa-Pro/NIb and the NIb/CP cleavage sites were different in P1 Sudan O.70 KF623541 (T in garlic isolates) and in P3’ (E in garlic isolates), a T, round bulb biotype; L, long bulb respectively. Concerning the analysis of the partial C biotype; Se, samples collected from terminal NIa-Pro protein, six residues (at positions onion fields for seed production. 8, 11, 12, 16, 17 and 32) of the 34 aa were conserved

Table 3. Range of nucleotide and amino acid sequence identities (%) in the studied genomic regions within OYDV-O popu- lations, OYDV-G populations, and between OYDV-O and OYDV-G. Comparisons were performed using all the Italian isolates and the Sudanese isolate from this study, and all available sequences for each compared region retrieved from the GenBank and reported in the phylogenetic trees.

Onion strain Garlic strain Between garlic and Within Italian onion strains Genomic region Between isolates Between isolates population

nt aa nt aa nt aa nt aa

NIa Pro-3’UTR 99.3–99.9 - 86.5–94.2 - 84.75–100 - 76.7–78.3 - NIa Pro-CP - 99.1–100 - 93.6–97.6 - 92.9–100 - 85.3–86.6 NIb 99.0–100 99.0–100 85.0–93.3 93.0–97.1 83.1–100 91.5–100 75.2–77.2 83.5–85.5 CP 99.2–100 98.4–100 87.5–96.9 93.8–100 88.2–99.9 86.8–100 79.0–87.5 81.3–96.5 3’UTR 99.0–100 - 94.7–98.6 - 87.5–100 80.0–90.5

442 Phytopathologia Mediterranea Molecular variability of Onion yellow dwarf virus from onion

Italy 152T Italy 155T Italy 5L Italy 6L.Se Italy 8T 51 Italy 13L.Se 84 Italy 2T.Se A. cepa 100 Italy 11L.Se 95 Italy 9L.Se 100 Italy 27T.Se 74 99 Italy 154T JX433020 Germany 100 JX433019 Argentina Sudan O.70 HQ258894 Australia

100 NC005029 China 100 NC 005029 China A. sativum 51 JN127342 Australia AB219833 Japan 100 AB219834 Japan NC007433 SYSV

0.05

Figure 2. Neighbour-joining tree based on multiple alignment of partial nucleotide sequences, including partial NIa-Pro, complete NIb and CP genes and 3’UTR, of OYDV between Italian and Sudanese isolates from this study, German and Argentine isolates from onion and isolates from garlic available from the database. The sequence of SYSV (NC007433) was used as an out-group. Bootstrap percentages of clades, reported along the branches of the tree with value >50%, derived from bootstrap-resampled data sets (1000 replications).

in OYDV-O and different from OYDV-G. At position tein 518 aa long, which contained all the conserved 10, the Sudanese and German isolates were different motifs CVDDFN, GNNSGQ, AMIEAWG, GQP- from the other onion isolates and produced the same STVVD, FTAAPIE and DGSRFDS identified and residue as the garlic isolates. used to detect members of the genus Potyvirus Phylogenetic analysis of the obtained 3’ terminal (Zheng et al., 2008, 2010). In addition, the putative nucleotide region of the 12 onion isolates from this active site of RNA-dependent RNA polymerase, study, the Argentine and German OYDV-O and six SG(X)3T(X)3NT(X)30GDD, was also conserved at the available OYDV-G (NC005029, JN127342, HQ258894, aa position 310–353 of the CP protein. When pairwise AJ510223, AB219833 and AB219834) produced a alignments of nt and aa sequences were performed, rooted tree that differentiated OYDV according to the Italian isolates showed up to 100% identity with the different host origins supported by 100% boot- each other, whereas they revealed an average iden- strap value. Of the onion isolates, the Italian OYDV- tity of 93.2% and 88.4% (nt) and 96.9% and 96.3% O were grouped together and were phylogenetically (aa) with the German and Argentine isolates from A. closer to the German isolate than the Argentine or cepa, respectively. The Sudanese OYDV isolate was Sudanese isolates (Figure 2). more distant from the Italian and German isolates than the Argentine isolate, with respective nt identi- NIb protein: variability and analysis ties of 85.2%, 85.3%, 88.9% and aa identities of 93.1%. 93.6%, and 96.3%. The NIb gene sequences of the Italian OYDV- Some residues differentiated the Italian OYDV- O isolates were 1554 nt long and encoded a pro- O isolates from the three foreign isolates, specifi-

Vol. 53, No. 3, December, 2014 443 A. Manglli et al.

Italy 5L Italy 9L.Se Italy 6L.Se Italy 155T Italy 152T Italy 11L.Se 60 Italy 154T A. cepa Italy 13L.Se 99 Italy 27T.Se

99 63 Italy 2T Italy 8T 100 JX433020 Germany JX433019 Argentina 88 Sudan O.70 HQ258894 Australia JN127342 Australia 100 100 AJ510223 China 83 A. sativum NC005029 China 57 AB219833 Japan 100 AB219834 Japan NC007433 SYSV

0.02

Figure 3. Neighbour-joining tree based on multiple alignment of complete NIb protein sequences of OYDV between Italian and Sudanese isolates from this study, German and Argentine isolates from onion and isolates from garlic available from the database. The sequence of SYSV (NC007433) was used as an out-group. Bootstrap percentages of clades, reported along the branches of the tree with value >50%, derived from bootstrap-resampled data sets (1000 replications).

cally at positions 79 (V instead of I), 279 (K instead the 15 isolates (11 from Italy and one from Sudan of of R/N/M), 338 (M instead of L/T/P), 343 (D in- this study and, three isolates from Argentina, Ger- stead of E) and 364 (E instead of G or K) of NIb gene. many and The Netherland retrieved from the Gen- Moreover, at least 22 conserved residues of different Bank) sharing identity values with each other’s polarities distinguished the onion isolates from the from a minimum of 87.5% to 100% at the nucleotide garlic isolates (data not shown) showing identity level (Table 3). The lowest value of this range was values from 75.2% to 77.2% at nucleotide levels and related to the Sudanese OYDV-O when compared from 83.5% to 85.5% at amino acid levels. The high- with Argentine, German and The Netherlands iso- est variability was located in the C terminus just lates, while this isolate showed an identity value of before the NIb/CP cleavage site. The phylogenetic approx. 89% with Italian isolates. The predicted CP analysis based on amino acid sequences (Figure 3), protein showed only a few differences at amino acid showed that onion isolates again grouped together levels in a few OYDV-O isolates compared to the ma- in a separate cluster from the garlic isolates, main- jority of the onion group (data not shown), where the taining a host grouping in the NIb coding region. Sudanese isolate was the most divergent from the other onion isolates with 95.3% of average aa identi- CP protein: variability and analysis ties due to 12 dissimilar residues, many of different polarities, mainly in the N-terminal region. The CP coding region of Italian OYDV-O isolates The putative CP protein of all OYDVs from onion was 771 nt long with a predicted protein of 257 aa. contained the aphid transmissibility aspartic acid- The CP coding region was highly conserved among alanine-glycine (DAG) triplet at the C-terminus of

444 Phytopathologia Mediterranea Molecular variability of Onion yellow dwarf virus from onion

Italy 8T Italy 9L.Se Italy 6L.se Italy 5L Italy 2T.Se Italy 155T A. cepa Italy 152T Italy 13L.Se Italy 11L.Se JX19433019 Argentina JX433020 Germany

94 99 Y11826 the Netherland Italy 27T.Se 77 Italy 154T A 71 Sudan O70 AB000836 the Netherlands A.sativum

66 GQ475374 Argentina A.sativum 98 GQ475377 Argentina A.sativum AF071226 Israel A.sativum 100 JN127344 Australia A.sativum AJ293278 China A.sativum FJ765739 China A.chinese 98 AB000840 Japan A.sativum 58 HM473189 Egypt A.sativum JN127343 Australia A.sativum GQ475390 Italy A. sativum 62 66 AJ409311 China A.sativum 100 GQ475360 USA A.sativum

99 AF228414 Brasil A.sativum 89 X89402 Argentina A.sativum DQ519034 India A.sativum AB551621 Myanmar A.sativum FR873734 India A.sativum

97 GQ475398 Italy A.sativum 96 GQ475410 Italy A.sativum

87 GQ475394 Italy A. sativum AB000837 Japan A.sativum 98 GQ475386 Argentina A.sativum

100 AB000841 Indonesia A.sativum 64 HQ258894 Australia A.sativum B

84 DQ925455 Vietnam A. porrum 83 JN127342 Australia A.sativum

89 AB000838 Japan A.sativum 50 AB219834 Japan A.sativum AJ409312 China A.sativum DQ925454 Vietnam A.porrum AJ307033 China A.sativum AJ292223 China A.sativum AJ409309 China A.sativum

52 AJ510223 China A.sativum 99 NC 005029 China A.sativum AB000843 Indonesia A.ascalonicum C 100 AB000845 Indonesia A.ascalonicum NC007433 SYSV

0.02

Figure 4. Neighbour-joining tree based on multiple alignment of complete CP protein sequences of OYDV between Italian and Sudanese isolates from this study, three onion isolates and 35 garlic isolates available from the database. The sequence of SYSV (NC007433) was used as an out-group. Bootstrap percentages of clades, reported along the branches of the tree with value >50%, derived from bootstrap-resampled data sets (1000 replications).

Vol. 53, No. 3, December, 2014 445 A. Manglli et al. the protein. Sequence similarities between OYDV- amino acid (D or E). In addition, at position 220 all O and OYDV-G were from 79% to 87.5% at the nt onion isolates revealed an A instead of a T which is level and from 81.3% to 96.5% at the aa level (Table conserved in all Allium isolates. 3). The variability amongst the isolates was in the N- Phylogenetic analysis based on the CP protein of terminus of the CP region which also produced the 15 onion, 30 garlic and five from differentAllium spp. greatest changes in aa residues in the predicted CP isolates indicated that OYDV isolates, a part from protein. Conversely, the residue at position 3 regard- two A. ascalonicum isolates, grouped into two main ing the NIb/CP cleavage site, as mentioned above, separate groups, in which the geographical origin was a neutral amino acid (P). This was conserved was not a discrimination character (Figure 4). The among the onion isolates but differed from almost onion isolates grouped together in group A but in a all of the other 35 Allium isolates showing an acidic separate clade close to three OYDV-G isolates from

Italy 8T Italy 13L.Se Italy 9L.Se Italy 154T Italy 27T.Se Italy 2T.Se Italy 155T A. cepa 60 Italy 6L.Se Italy 11L.Se A 73 Italy 152T Italy 5L JX433019 Argentina 59 Sudan O.70 74 JX433020 Germany 68 AB000836 the Netherlands A.sativum X89402 Argentina A. sativum AB000843 Indonesia A. ascalonicum 95 AB000845 Indonesia A. ascalonicum DQ519034 India A. sativum 53 AJ409311 China A. sativum HQ258894 Australia A. sativum 78 AB000841 Indonesia A. sativum AB000837 Japan A. sativum

91 DQ925455 Vietnam A. porrum JN127342 Australia A. sativum B AJ409309 China A. sativum DQ925454 Vietnam A. porrum AJ409312 China A. sativum AB000838 Japan A sativum AJ292223 China A. sativum AJ307033 China A. sativum 60 AJ510223 China A. sativum 98 NC005029 China A. sativum AJ293278 China A. sativum FJ765739 China A. chinese C 99 HM473189 Egypt A. sativum 55 AB000840 Japan A. sativum NC007433 SYSV

0.05

Figure 5. Neighbour-joining tree based on multiple alignment of 3’UTR sequences of OYDV between Italian and Sudanese isolates from this study, Argentine and German onion isolates and 23 Allium spp. isolates available from database. The sequence of SYSV (NC007433) was used as an out-group. Bootstrap percentages of clades, reported along the branches of the tree with value >50%, derived from bootstrap-resampled data sets (1000 replications).

446 Phytopathologia Mediterranea Molecular variability of Onion yellow dwarf virus from onion non-Asian countries, which shared an aa homology Discussion of 95–96%. The Argentine OYDV-O showed the near- Although OYDV was firstly identified in onion est genetic evolutionary relationship with Italian (Melhus et al., 1929), studies on onion isolates have isolates. On the other hand, OYDV-O70 from Sudan been mainly restricted to diagnosis and disease ef- was clearly distant from all the other onion isolates. fects on the crops; moreover, only two complete The phylogenetic tree showed low bootstrap values (the Argentine and German isolates) and one partial (<50%) for the major branches. However, the analy- (the Netherland isolate) sequences have been de- sis using the Maximum Parsimony method retained posited in Genbank. On the contrary, many studies the sub-grouping as reported above. have been carried out on OYDV from infected gar- lic (OYDV-G) focusing on the characterization and 3’UTR variability and analysis phylogenetic analysis of partial or complete genome of the virus isolates mainly from Asia but also from The 3’ UTR sequences of isolates from Italy were Argentina, Africa and Europe including Italy (Shibo- almost all identical, with the exception of two iso- leth et al., 2001; Arya et al., 2006; Baghalian et al., 2010; lates which differed by only one nucleotide. In ad- Parrano et al., 2012; Soliman et al., 2012; Mohammed dition, they were more similar to the OYDV iso- et al., 2013). late from Argentina (98.6%) and slightly dissimilar Our data extend existing knowledge since sev- (92.2%) from Sudanese and German isolates. The eral OYDV-O isolates were partially sequenced and 3’ UTR sequences of all OYDV isolates from onion examined. Firstly, the comparison of the 3’ terminal contained 3 or 5 nt less among CP 37-38 residues 2637 nt region of the genome, encompassing the par- than the corresponding sequences of the majority tial NIa-Pro, the complete NIb and CP genes and of OYDV-G isolates retrieved from the GenBank. 3’UTR, of the 11 OYDV-O Italian isolates revealed Between OYDV-O and OYDV-G, nucleotide iden- a high nucleotide and amino acid sequence identity tity values in the 3’UTR region ranged from 80% to each other (Table 3). This homogeneous population 90.48% (Table 3). is undoubtedly due to the same geographic origin of Phylogenetic analysis of the 3’ UTR confirmed the isolates (the same cultivar grown in the same IGP three major groups (Figure 5) with few differences protected area during a limited period of survey). from the CP phylogenetic tree. In A, all onion iso- In the past, OYDV-O was detected in Italy but the lates were grouped on the evolution linkage of two isolates were not sequenced (Marani and Bertaccini, OYDV-G from the Netherlands and Argentina. 1983; Dovas and Vovlas, 2003; Parrella et al., 2005) and during our previous studies no more Italian Recombination analysis isolates were found in regions other than Calabria (data not shown). Consequently, molecular variabil- Recombination detection analysis of the complete ity analysis amongst isolates from different Italian 2427 nucleotide sequences gave one significant event. regions was not possible. This event shows recombination between the Italian It was interesting to include in our study the geo- genotype (in particular OYDV 152T with 95.3% simi- graphically distant Sudanese isolate (OYDV-O.70) larity) as major parent and the Sudanese genotype which showed significant differences in nucleotide (90.7% similarity) as the minor parent, which led and amino acid sequences when compared to the to a recombinant isolate identified in the Argentine Italian onion isolates showing a nt and aa identities isolate. In this recombination event, a region (748- not more than 89% and 94.7%, respectively. When 1626 nt of the analyzed nucleotide sequence) of the the phylogenetic relationship was extend to the oth- Argentine isolate was replaced with the core and 3’ er three available OYDV-O, the analysis of the 3’ nt terminal NIb region of the Sudan isolate. A high de- terminal region, the NIb and CP proteins revealed gree of confidence was shown by six recombination that the Sudanese isolate is an ancestor of the other detection methods implemented in RDP 4 program onion isolates (Figures 2, 3 and 4) with a closer re- (i.e. average P-value of 5.035 × 10-7 by RDP; 3.673 × lationship to the Argentine isolate only in the NIb 10-6 by BOOTSCAN; 5.287 × 10-11 by Maximum ChiS- region (Figure 3). quare; 5.354 × 10-12 by Chimera; 6.509 × 10-11 by Sister The recombination analysis explained the differ- Scan; 1.983 × 10-10by 3Seq). ent position of the Argentine isolate in the phyloge-

Vol. 53, No. 3, December, 2014 447 A. Manglli et al. netic trees generated from different regions, showing sites between OYDV-O and OYDV-G, are frequent that the recombination event with the Sudanese iso- in many potyvirus species (http://dpvweb.net/po- late as minor parent occurred in the NIb region. This tycleavage/species.html). These do not always sup- first recombinant study of OYDV-O provides the port species demarcation within the genus Potyvirus first important context in the virus evolution, and (Adams et al., 2005a). motivates further research within the species. Moreover, even if OYDV-O and OYDV-G are host- In our study, the Italian and the Sudanese OY- specific as onion isolates cannot infect garlic and leek DV-O isolates were also compared with the corre- whereas garlic isolates infects shallot, leek and scal- sponding published sequences of the isolates from lion but not onion (Katis et al., 2012; Celli et al., 2013), different Allium spp. This analysis revealed that nu- this biological performance between strains of the cleotide identity in the NIb/CP/3’UTR region was same viral species is quite frequent in plant viruses greater in the maximum average value (85.0%) than (i.e., potyviruses). This is controlled by a few amino 81.5% reported by Celli et al. (2013), who suggested acid mutations in genes involved in virus-host inter- that OYDV-O and OYDV-G might be different spe- actions and long distance movement (Boevink and cies. Equally, when NIb, CP and 3’UTR regions were Oparca, 2005; Lopez-Moya et al., 2009). separately analyzed for similarity to garlic isolates, There are not enough data (complete genomes) the greater number of onion isolates determined an on OYDV-O from the Middle East and Central Asia, increase in the minimum and maximum identity val- the probable regions of origin of A. cepa (Messiaen ues (Table 3). and Rouamba, 2004), to assume different ancestors Our data limited to the investigated region there- for the two OYDV strains. In any case, the world ex- fore indicate that onion and garlic isolates could still change of vegetative materials and the cultivation in be considered strains of the same species, according the same area of different Allium species has prob- to the proposed thresholds for species demarcation ably favoured mixed populations or recombinant in the Potyviridae family (NIb: 75%; CP: 76%; 3’UTR: isolates in many countries, since geographical origin 76%) (Adams et al., 2005b). is not a demarcation criterion in OYDV phylogenetic When phylogenetic analysis of OYDV was per- grouping (Figures 4 and 5). formed, the separation of isolates into onion and gar- In conclusion, this study and the recent genetic lic groups was only obtained when the 3’ terminal nt analysis of Celli et al. (2013) highlight the need for region (partial NIa-Pro/NIb/CP/3’UTR) and NIb more extensive investigations to better clarify the tax- protein trees were constructed (Figures 2 and 3). On onomic relationships between OYDV isolates origi- the contrary, the phylogenetic trees processed in the nating from different Allium spp. hosts. It would be CP gene and 3’UTR (Figures 4 and 5) which included very worthwhile to examine more OYDV-O isolates greater numbers of OYDV-G sequences from A. sati- from other countries, mainly from the geographical vum, A. chinense, A. porrum and A. ascalonicum, showed areas where the A. cepa is presumed to have origi- that the onion isolates were not grouped separately nated as well as from countries where this crop is from OYDV-G. Onion isolates were phylogenetically widely grown. Despite the failure in finding other close to OYDV-G originating in non-Asian countries Italian isolates from regions other than Calabria, along the linkage of OYDV-G from the Netherlands where the typical ‘Rossa di Tropea’ onion is grown, (AB000836) and Argentina (GQ475374; GQ475377; the foreign isolate from Sudan enabled us to iden- X89402) sharing with them nt identity values from tify the recombination event amongst the OYDV-O 83–87%. In the light of these results and according and some molecular specificity of these isolates com- to the convention statement of the 9th ICTV Report pared to those of garlic isolates (Parrano et al., 2012). on species demarcation criteria over whole genomes Further research is now underway aimed at obtain- (< 76%) and in the CP protein (<80%) in the family ing complete sequences of the Italian OYDV isolates Potyviridae (King et al., 2011), OYDV-O and OYDV-G and collecting more virus isolates. might again be considered strains of the same virus and not two different virus species. Acknowledgements In accordance with this strain differentiation, substitutions of one residue at polyprotein cleav- This study was supported, in part, by grants age sites, as observed at NIa-Pro/NIb and NIb/CP from Regione Calabria, Italy, Accordo di Program-

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Accepted for publication: 14 May, 2014 Published online: December 22, 2014

450 Phytopathologia Mediterranea